The Structure and Function of a Microbial Allantoin Racemase Reveal the Origin and Conservation of a Catalytic Mechanism.

The S enantiomer of allantoin is an intermediate of purine degradation in several organisms and the final product of uricolysis in nonhominoid mammals. Bioinformatics indicated that proteins of the Asp/Glu racemase superfamily could be responsible for the allantoin racemase (AllR) activity originally described in Pseudomonas species. In these proteins, a cysteine of the catalytic dyad is substituted with glycine, yet the recombinant enzyme displayed racemization activity with a similar efficiency (kcat/KM ≈ 5 × 104 M-1 s-1) for the R and S enantiomers of allantoin. The protein crystal structure identified a glutamate residue located three residues downstream (E78) that can functionally replace the missing cysteine; the catalytic role of E78 was confirmed by site-directed mutagenesis. Allantoin can undergo racemization through formation of a bicyclic intermediate (faster) or proton exchange at the chiral center (slower). By monitoring the two alternative mechanisms by 13C and 1H nuclear magnetic resonance, we found that the velocity of the faster reaction is unaffected by the enzyme, whereas the velocity of the slower reaction is increased by 7 orders of magnitude. Protein phylogenies trace the origin of the racemization mechanism in enzymes acting on glutamate, a substrate for which proton exchange is the only viable reaction mechanism. This mechanism was inherited by allantoin racemase through divergent evolution and conserved in spite of the substitution of catalytic residues.

Gene context analysis reveals functional divergence between hypothetically equivalent enzymes of the purine-ureide pathway.

A major problem of genome annotation is the assignment of a function to a large number of genes of known sequences through comparison with a relatively small number of experimentally characterized genes. Because functional divergence is a widespread phenomenon in gene evolution, the transfer of a function to homologous genes is not a trivial exercise. Here, we show that a family of homologous genes which are found in purine catabolism clusters and have hypothetically equivalent functions can be divided into two distinct groups based on the genomic distribution of functionally related genes. One group (UGLYAH) encodes proteins that are able to release ammonia from (S)-ureidoglycine, the enzymatic product of allantoate amidohydrolase (AAH), but are unable to degrade allantoate. The presence of a gene encoding UGLYAH implies the presence of AAH in the same genome. The other group (UGLYAH2) encodes proteins that are able to release ammonia from (S)-ureidoglycine as well as urea from allantoate. The presence of a gene encoding UGLYAH2 implies the absence of AAH in the same genome. Because (S)-ureidoglycine is an unstable compound that is only formed by the AAH reaction, the in vivo function of this group of enzymes must be the release of urea from allantoate (allantoicase activity), while ammonia release from (S)-ureidoglycine is an accessory activity that evolved as a specialized function in a group of genes in which the coexistence with AAH was established. Insights on the active site modifications leading to a change in the enzyme activity were provided by comparison of three-dimensional structures of proteins belonging to the two different groups and by site-directed mutagenesis. Our results indicate that when the neighborhood of uncharacterized genes suggests a role in the same process or pathway of a characterized homologue, a detailed analysis of the gene context is required for the transfer of functional annotations.

Biochemical characterization of mouse d-aspartate oxidase.

D-amino acids research field has recently gained an increased interest since these atypical molecules have been discovered to play a plethora of different roles. In the mammalian central nervous system, d-aspartate (D-Asp) is critically involved in the regulation of glutamatergic neurotransmission by acting as an agonist of NMDA receptor. Accordingly, alterations in its metabolism have been related to different pathologies. D-Asp shows a peculiar temporal pattern of emergence during ontogenesis and soon after birth its brain levels are strictly regulated by the catabolic enzyme d-aspartate oxidase (DASPO), a FAD-dependent oxidase. Rodents have been widely used as in vivo models for deciphering molecular mechanisms and for testing novel therapeutic targets and drugs, but human targets can significantly differ. Based on these considerations, here we investigated the structural and functional properties of the mouse DASPO, in particular kinetic properties, ligand and flavin binding, oligomerization state and protein stability. We compared the obtained findings with those of the human enzyme (80% sequence identity) highlighting a different oligomeric state and a lower activity for the mouse DASPO, which apoprotein species exists in solution in two forms differing in FAD affinity. The features that distinguish mouse and human DASPO suggest that this flavoenzyme might control in a distinct way the brain D-Asp levels in different organisms.

Distribution of Hydrogenases in Cyanobacteria: A Phylum-Wide Genomic Survey.

Microbial Molecular hydrogen (H2) cycling plays an important role in several ecological niches. Hydrogenases (H2ases), enzymes involved in H2 metabolism, are of great interest for investigating microbial communities, and producing BioH2. To obtain an overall picture of the genetic ability of Cyanobacteria to produce H2ases, we conducted a phylum wide analysis of the distribution of the genes encoding these enzymes in 130 cyanobacterial genomes. The concomitant presence of the H2ase and genes involved in the maturation process, and that of well-conserved catalytic sites in the enzymes were the three minimal criteria used to classify a strain as being able to produce a functional H2ase. The [NiFe] H2ases were found to be the only enzymes present in this phylum. Fifty-five strains were found to be potentially able produce the bidirectional Hox enzyme and 33 to produce the uptake (Hup) enzyme. H2 metabolism in Cyanobacteria has a broad ecological distribution, since only the genomes of strains collected from the open ocean do not possess hox genes. In addition, the presence of H2ase was found to increase in the late branching clades of the phylogenetic tree of the species. Surprisingly, five cyanobacterial genomes were found to possess homologs of oxygen tolerant H2ases belonging to groups 1, 3b, and 3d. Overall, these data show that H2ases are widely distributed, and are therefore probably of great functional importance in Cyanobacteria. The present finding that homologs to oxygen-tolerant H2ases are present in this phylum opens new perspectives for applying the process of photosynthesis in the field of H2 production.

Presence and impact of allelic variations of two alternative s-kdr mutations, M918T and M918L, in the voltage-gated sodium channel of the green peach aphid Myzus persicae.

BACKGROUND: Pyrethroids have been widely employed in order to control several agricultural pests, including Myzus persicae. Target-site resistance is the main mechanism that confers insensitivity to this class of compounds, and the most common amino acid substitutions are kdr (L1014F) and s-kdr (M918T), but recently another mutation in the s-kdr locus (M918L) has been described in French and Korean populations of M. persicae. RESULTS: Molecular analysis of several Italian populations of M. persicae by pyrosequencing revealed the presence of the new s-kdr mutation (M918L) in different forms. It was found in two different nucleotide polymorphisms (a/t or a/c substitution), in heterozygous or homozygous status, and also in combination with the classic kdr and s-kdr. Bioassays on populations carrying the M918L mutation show that it strongly affects pyrethroid efficacy, particularly of type II pyrethroids such as lambda-cyhalothrin, while it has no effect against DDT. CONCLUSION: This work provides more information about the new s-kdr M918L mutation in M. persicae, describing a more complicated situation arising from the possible combination with the classic L1014F and M918T. Our data open new questions concerning the origin of these new genotypes with different combinations of target-site mutations, and also their possible influence on control strategies.

Ureidoglycolate hydrolase, amidohydrolase, lyase: how errors in biological databases are incorporated in scientific papers and vice versa.

An opaque biochemical definition, an insufficient functional characterization, an interpolated database description, and a beautiful 3D structure with a wrong reaction. All these are elements of an exemplar case of misannotation in biological databases and confusion in the scientific literature concerning genes and enzymes acting on ureidoglycolate, an intermediate of purine catabolism. Here we show biochemical evidence for the relocation of genes assigned to EC 3.5.3.19 (ureidoglycolate hydrolase, releasing ammonia), such as allA of Escherichia coli or DAL3 of Saccharomyces cerevisiae, to EC 4.3.2.3 (ureidoglycolate lyase, releasing urea). The EC 3.5.3.19 should be more appropriately named ureidoglycolate amidohydrolase and include genes equivalent to UAH of Arabidopsis thaliana. The distinction between ammonia- or urea-releasing activities from ureidoglycolate is relevant for the understanding of nitrogen metabolism in various organisms and of virulence factors in certain pathogens rather than a nomenclature problem. We trace the original fault in database annotation and provide a rationale for its incorporation and persistence in the scientific literature. Notwithstanding the technological distance, yet not surprising for the constancy of human nature, error categories and mechanisms established in the study of the work of amanuensis monks still apply to the modern curation of biological databases.

Chemical basis of nitrogen recovery through the ureide pathway: formation and hydrolysis of S-ureidoglycine in plants and bacteria.

While some organisms, including humans, eliminate oxidized purines to get rid of excess nitrogen, for many others the recovery of the purine ring nitrogen is vital. In the so-called ureide pathway, nitrogen is released as ammonia from allantoate through a series of reactions starting with allantoate amidohydrolase (AAH), a manganese-dependent enzyme found in plants and bacteria. We report NMR evidence that the true product of the AAH reaction is S-ureidoglycine, a nonstandard alpha-amino acid that spontaneously releases ammonia in vitro. Using gene proximity and logical genome analysis, we identified a candidate gene (ylbA) for S-ureidoglycine metabolism. The proteins encoded by Escherichia coli and Arabidopsis thaliana genes catalyze the manganese-dependent release of ammonia through hydrolysis of S-ureidoglycine. Hydrolysis then inverts the configuration and yields S-ureidoglycolate. S-Ureidoglycine aminohydrolase (UGHY) is cytosolic in bacteria, whereas in plants it is localized, like allantoate amidohydrolase, in the endoplasmic reticulum. These findings strengthen the basis for the known sensitivity of the ureide pathway to Mn availability and suggest a further rationale for the active transport of Mn in the endoplasmic reticulum of plant cells.